N-protected .alpha.-L-aspartyl-L-phenylalanine methyl ester, such as in particular N-benzyloxycarbonyl-.alpha.-L-aspartyl-L-phenylalanine methyl ester, is an important precursor of the "intense sweetener" aspartame, a product having a sweetening power of approximately 200 times that of sucrose and with an excellent taste profile without, for example, the bitter aftertaste of other intense sweeteners such as, for example, saccharin and cyclamate. The sweetener aspartame is used, inter alia, in a wide range of products such as soft drinks, sweets, "table-top sweeteners", pharmaceuticals, etc.
Various methods are known for the preparation of aspartame. In addition to chemical preparation methods there are also enzymatic preparation methods, which owe their importance primarily to the fact that enzymatic coupling takes place in a stereoselective and regioselective manner. Enzymatic L,L-coupling of N-protected aspartic acid, in particular of N-benzyloxycarbonyl-aspartic acid (hereinafter also designated by Z-Asp), and L- (or DL-) phenylalanine methyl ester, or of acid salts derived therefrom such as, for example, the hydrochloride salt (in the following also designated by PM), with formation of an N-protected aspartame precursor, has been thoroughly studied and described to date. An overview of aspartame preparation methods is given by K. Oyama in Chapter 11 (pp. 237-247) of "Chirality in Industry", John Wiley & Sons Ltd., 1992.
The enzymatic coupling reaction in question, which as a rule is carried out at a pH of from 6 to 7.5 in the presence of a neutral protease, in particular of a metallo-protease such as, for example, thermolysin, is an equilibrium-controlled reaction. In order to achieve high degrees of conversion in such enzymatic coupling reactions, specific measures are necessary according to the state of the art. Thus, for example, U.S. Pat. No. 4,165,311 (which is regarded to be the nearest state of the art) makes use of the fact that the equilibrium in the coupling reaction can be shifted to the right by the formation of a precipitating addition compound of N-protected aspartame, in particular of N-benzyloxycarbonyl-.alpha.-L-aspartyl-L-phenylalanine methyl ester (hereinafter also designated by Z-APM), with D- or L-phenylalanine methyl ester present in the reaction mixture. Such addition compounds of the aspartame precursor are also designated by Z-APM.D-PM or Z-APM.L-PM, respectively. In order to form such addition products it is desirable, according to the state of the art, for the coupling reaction of Z-Asp and L-PM to be carried out with at least double the molar quantity of L-PM with respect to Z-Asp, or in the presence of an at least equivalent amount of D-PM in order to achieve high degrees of conversion, i.e. &gt;60%, preferably &gt;80% based on Z-Asp. In practice, these enzymatic coupling reactions are therefore usually described in ratios of PM to Z-Asp of, for example, from 2.0 to 2.5:1 or higher. Although with such embodiments high degrees of conversion to the desired product are indeed achieved, these methods have a number of drawbacks, viz.:
(a) handling and further processing of the precipitate in order to obtain the ultimately desired aspartame (hereinafter: APM) is laborious, partly because the addition product is relatively difficult to filter and must be washed thoroughly in order to obtain APM which contains only small amounts of impurities; PA0 (b) recovery and/or recirculation is necessary of the component(s) present in excess and of the non-APM component to be liberated from the precipitated addition product of the coupling product; if the coupling reaction is carried out with DL-PM, the remaining D-PM should, as a rule, when being processed, also be racemized, as a rule via DL-phenylalanine. These methods are therefore less suitable for application on a commercial scale.
It should be noted that WO-A-92/02617 describes an enzymatic coupling reaction of virtually equal amounts of Z-Asp and L-PM.HCl (in a molar ratio of approximately 1.2:1) in an aqueous medium and in the presence of acetic acid at pH=7. In this case, use is made of protease enzyme crystals immobilized by cross-linking, but the degree of conversion achieved is only approximately 20%. EP-A-0149594 describes the use of formyl-Asp (F-Asp) for an enzymatic coupling reaction in an aqueous medium in a 1:1 ratio of F-Asp to L-PM. However, because of the formation of the F-APM.L-PM addition product, the conversion of F-Asp remains distinctly below 50%, and the yield achieved in the process is found to be very low (approximately 12% after processing to give F-APM).
In the same way the article of Zhou F. et al. (in: Huaxue Fanying Goncheng Yu Gongyi, 1992, 8(4), pp 413-419 (in Chinese); abstract in Chemical Abstracts, 120 (no. 35), 31-1-94, abstract 48817v) describes amongst other things an experiment of 1:1 enzymatic coupling of Z-L-asp and L-PM at an initial pH of 6. However, also under these conditions, due to the usual formation of the ZAPM.PM addition product the conversion of Z-Asp is at best 46.1%. It should be noted that the L-PM conversion (of at most 92.2%) as reported by Zhou F. et al. accounts for the sum of the (chemical) coupling of L-PM into Z-APM and the concomittant precipitation of one equivalent of L-PM with Z-APM. There is no teaching in this article that chemical conversion of L-PM above 50% can be reached.
It should also be noted, incidentally, that the esters present in the coupling reaction system are relatively sensitive to chemical hydrolysis. Thus, PM is hydrolyzed to form phenylalanine (hereinafter also designated by Phe); Z-APM is hydrolyzed to form Z-protected aspartylphenylalanine (sometimes designated by Z-AP). This undesirable side reaction occurs especially at a pH &gt;6, or &gt;4, and is stronger the more the pH deviates from said values and the residence time under reaction conditions is longer.
Until now it has been generally assumed that in the enzymatic coupling of Z-Asp and L-PM, starting from equivalent or virtually equivalent amounts of Z-Asp and L-PM without the presence of a corresponding amount of D-PM, or without taking other measures to shift the coupling equilibrium, no conversions greater than 50%, calculated on the basis of Z-Asp, could be achieved. As far as the enzymatic coupling in an aqueous medium is concerned, Zhou and Huang (Indian J. Chem., 32B, pp 35-39, 1993) stated even recently that the optimum conditions for the reaction, using immobilized protease, are at a ratio of Z-Asp to PM of 1:4. It should be noted in this context that, when an immobilized protease is used (cf., e.g. Biotechnology, 3, pp. 459-464, 1985; Nakanishi et al.) much of the product formed is absorbed in the resin employed fox immobilization and has to be removed therefrom via a separate extraction step. The results achieved by Nakanishi et al. with a 1:1 ratio of Z-Asp to L-PM in an aqueous medium, a yield of at most 58% at a relatively low concentration (80 mM), are therefore irrelevant to industrial practice. Moreover, at such low concentrations, often used for determining initial reaction rates, coupling reaction proceeds without formation of a precipitate.
Alternative ways of shifting the coupling equilibrium have been described in, inter alia, (i) J. Org. Chem. 46, p. 5241 (1981): use of an immobilized protease and an organic solvent not miscible with water; similarly, JP-B-8533840, where yields of only approximately 20-30% are shown when use is made of 1:1 molar ratios; (ii) GB-A-2250023: use of immobilized protease and water-miscible organic solvent; similarly EP-A-0272564 in acetonitrile, where, while it is suggested that the ratio of N-protected Asp to L-PM can be between 10:1 and 1:10, the examples show, nevertheless, that only a considerable excess of L-PM is being considered and that in the case of stoichiometric or virtually stoichiometric ratios poor conversions and yields are obtained. Stoichiometric ratios are also called equimolar ratios. From the examples described in GB-A-2250023 it can likewise be seen, incidentally, that higher yields are achieved the higher the ratio of L-PM to N-protected Asp (at 2:1, the yield is approximately 85%, at 1:1 only approximately 50%). In such alternative embodiments, the shift of the equilibrium is not achieved by a precipitate being formed, but rather by the coupling product formed being transferred to the organic phase. Apart from cost-increasing aspects as a result of often unavoidable solvent losses when using organic solvents, another drawback of such alternative embodiments is that, during processing to produce aspartame, special measures must be taken to remove the organic solvents used in the coupling reaction. When adding (or carrying out the reaction in, or in the presence of, as the case may be) an organic solvent such as, for example, acetonitrile or dimethyl formamide, or substances such as di- and triglyme (see EP-A-0278190), as a rule only low yields of Z-APM and the like are achieved, unless the reaction is carried out at a high molar ratio of L-PM (.HCl) to Z-Asp.
It should additionally also be noted that it is not unusual, in the case of chemical coupling reactions (starting from N-protected aspartic anhydride, for example the N-formyl derivative, and L-Phe or L-PM), for the reaction to take place at stoichiometric or virtually stoichiometric ratios of the reactants, but this teaches nothing concerning enzymatic coupling reactions using Z-Asp as a starting material in water.
Attention should, however, be drawn to DE-A-3517361, which discloses, for an enzymatic coupling reaction, that the reactants Z-Asp and L-PM may indeed be present in virtually stoichiometric ratios, but--for adduct formation--(and instead of the minimum equivalent excess required of L-PM or D-PM according to the state of the art cited hereinabove) an at least equivalent amount of an organic amine compound is employed, in which at least one C.sub.6 hydrocarbon radical is present. In practice, such a method is of little relevance for the preparation of APM because, on the one hand, the addition product formed has to be cleaved by acidification, liberating the amine, and, on the other hand, a further organic component "foreign to the process" is introduced which, in recirculation and filtrate streams of the process, is difficult to separate from the starting materials used for the APM synthesis.
There was therefore a need for simple and efficient, preferably stoichiometric, enzymatic coupling of Z-Asp and L-PM, which affords both high conversion and low consumption of starting materials and a limitation in recycling streams, and yields a readily filterable product without the need for the presence of at least equivalent amounts of D-PM (or an additional equivalent of L-PM) or the like, and without the necessity of adding organic solvents or amines in the coupling reaction.